Novel inhibitors against Cryptosporidium parvum through blocking its energy metabolism

Through systematic structural optimization and modification of anti-Cryptosporidium parvum compound NSC158011 (N-1-naphthalenyl-2-(phenylthiol) ethane thioamide), we constructed a virtual compound library comprising 1,440 derivatives, and 29 derivatives were selected via molecular docking for chemical synthesis. Among them, eight derivatives exhibited significant inhibitory effects on LDH compared with NSC158011. In vitro examination found that two derivatives could markedly reduce the number of C. parvum in cells while exhibiting reduced toxicity. In vivo studies in model animals (IFN-γ knockout mouse) and native animals (calves) further validated the derivatives' potent anti-parasitic effects and their ability to alleviate intestinal villus atrophy caused by parasitic infections. Further investigations revealed that compound treatment was associated with upregulated expression of intestinal tight junction proteins (ZO-1, Occludin, Claudin-1) and downregulated the expression level of inflammatory protein Caspase-1. Analysis of the gut microbiota and metabolomics in calves indicated that treatment significantly restored the abundance of beneficial flora and regulated key metabolites, including isocitric acid and ornithine. The two derivatives identified in this study represent promising lead compounds for further development as anti-cryptosporidiosis agents, with potential relevance to livestock production and public health.